Friedreich's ataxia is a severe autosomal recessive neurodegenerative disease, the most frequent inherited ataxia in Caucasians. It is caused by transcriptional silencing of the FXN gene induced by expansion mutation of the GAA repeats located in the first intron of this gene. Importantly, coding sequence of the FXN gene is intact and fully capable of expressing functional frataxin. Friedreich's ataxia patients homozygous for GAA expansion have very low frataxin mRNA and protein levels when compared with heterozygous carriers and healthy controls. Currently there is no effective treatment for Friedreich's ataxia. Thus far, a very limited number of compounds have been shown to alleviate GAA-induced transcriptional silencing to a small extent in vitro. The objective of this project is to design, construct and characterize a high-throughput screening strategy aimed towards discovering new pharmacological probes capable to stimulate gene expression blocked by pathologically expanded GAA repeats. To achieve these goals we propose the following specific aims: (i) Development of a cell-based reporter assay for identification of compounds capable to alleviate GAA repeats-induced transcriptional silencing. We will construct and test the reporter green fluorescent protein (GFP) minigene containing long intronic GAA repeats. Our preliminary experiments showed a significant silencing of this reporter gene by a tract of 560 GAA repeats. In order to improve the signal-to-background ratio of the screen, a longer GAA repeat tract will be introduced to the reporter. Additionally, the endogenous frataxin promoter will be used to express the reporter minigene. Experiments will be conducted to ensure that the silencing of the GFP reporter gene is mimicking transcriptional inhibition of the FXN gene. We will also develop selectivity assay using an independent reporter red fluorescent protein (RFP) minigene without GAA repeats. (ii) Configuration of the assay for a high-throughput screen. A counter-screen strategy based on firefly luciferase reporter containing long intronic GAA repeats will be developed. Additionally, follow-up protocol involving lymphoblastoid cell lines from Friedreich's ataxia patients will be developed to verify specificity of hits towards FXN gene induction. We will also conduct a pilot screen using NIH Clinical Collection library of compounds. Collectively, these studies will result in the development of the HTS assay ready for implementation into the screening program at the Molecular Libraries Probe Production Centers Network. Furthermore, we will propose a strategy of lead development in the follow-up research program designed to discover new therapeutic probes for Friedreich's ataxia and perhaps other diseases caused by transcriptional silencing. PUBLIC HEALTH RELEVANCE: This project can lead to the discovery of new compounds with a therapeutic activity towards Friedreich's ataxia, the most common inherited ataxia. Results of this study may also be applicable in the development of innovative therapeutic approaches for Fragile X syndrome and other neurological diseases as well as cancer.